Project Summary Cardiovascular disease is the leading cause of death worldwide. It is a multifaceted chronic inflammatory disease characterized by the accumulation of modified lipoproteins and immune cells in the aorta, vascular dysfunction, and low-grade chronic inflammation. Despite the strides that have been made in the past decade, there is still much that is unknown about the mechanisms behind disease progression. The uptake of modified low density lipoproteins (mLDL) by macrophages has been well characterized as being a major mechanism in the development of atherosclerosis. Our laboratory show that B cells can uptake mLDL and this uptake induces some significant phenotypical changes in B cells and upregulation of LXR-dependent targets. While mechanisms and role of mLDL uptake by M?s are well-characterized, the pathways of mLDL uptake in B cells are unclear and nothing is yet known about the impact of mLDL uptake on B cell functions. Our preliminary data suggest that acLDL uptake by B cells induces a unique gene profile, which supports inhibitory pathways including GRAIL. GRAIL is a well-known regulator for T-cell tolerance and T cell activation. Our data demonstrate that GRAIL is also expressed in B cells and play an important role in B cell activation and B cell anergy. Importantly, B cell-specific deficiency results in increased atherosclerosis. In Aim 1 of this application, we propose to test molecular mechanisms whereby GRAIL expression and mLDL uptake control B cell activation in atherosclerosis. First, we will test mechanisms of mLDL uptake by B cells using SR-B1, MARCO, CXCL16, and CD163 deficient mice or blocking Abs BCR-transgenic and Fc?RIIB- deficient mice. We will test how mLDL uptake induces some inhibitory pathways in B cells with the focus on LXR-mTOR-Otub1-GRAIL axis. Next, we will determine pathways by which GRAIL shapes B cell activation, investigate GRAIL-dependent BCR signaling, B cell survival and IL-4-dependent signaling in B cells, and examine the role of B cell specific GRAIL in atherosclerosis. In Aim 2, we will determine how atherosclerosis affects B cell anergy and test a role of GRAIL and mLDL uptake in stability and functions of anergic B cells. We will compare phenotypes of anergic B cells from young, aged Apoe-/- mice, and age-matched naturally occurring anergic BL/6 B cells. We will also test how atherosclerosis affects stability of anergic B cells in adoptive transfer experiments using Ars/A1 transgenic mice, a model of B cell anergy. Next, we will investigate how GRAIL affects B cell anergy and atherogenesis using Ars/A1 mice that would receive AAV?shRNA-Rnf128 under B cell specific promoter and AAV-PSCK9. The implications from this work are significant since the results will provide the first evidence for a role of mLDL uptake in B cell biology via GRAIL-dependent regulation of B cell anergy and B cell activation in atherosclerosis.